Human zinc binding proteins

ABSTRACT

The present invention provides three zinc binding proteins (designated individually as ZB-1, ZB-2, and ZB-3, and collectively as ZB) and polynucleotides which identify and encode ZB. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding ZB and a method for producing ZB. The invention also provides for use of ZB and agonists, antibodies, or antagonists specifically binding ZB, in the prevention and treatment of diseases associated with expression of ZB. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding ZB for the treatment of diseases associated with the expression of ZB. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding ZB.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of zincbinding proteins and to the use of these sequences in the diagnosis,prevention, and treatment of diseases related to disregulated cellgrowth and proliferation including cancer.

BACKGROUND OF THE INVENTION

Zinc binding (ZB) domains are found in numerous proteins which areinvolved in protein-nucleic acid or protein-protein interactions. ZBproteins are commonly involved in the regulation of gene expression, andmay serve as transcription factors and signal transduction molecules. AZB domain is generally composed of 25 to 30 amino acid residues whichform one or more tetrahedral ion binding sites. The binding sitescontain four ligands consisting of the sidechains of cysteine, histidineand occasionally aspartate or glutamate. The binding of zinc allows therelatively short stretches of polypeptide to fold into definedstructural units which are well-suited to participate in macromolecularinteractions (Berg, J. M. et al. (1996) Science 271:1081-1085).

Classes of ZB domains are characterized according to the number andpositions of the residues involved in the zinc atom coordination. ZBdomains of the C₂ H₂ type were first identified in the proteintranscription factor IIIA (TFIIIA; Hanas, J. et al. (1983) J. Biol.Chem. 258:14120-14125) and represent the most abundant DNA binding motifin eukaryotic transcription factors (Berg, supra). These domains, alsoknown as "zinc fingers", are characterized by tandem arrays of sequencesthat approximate the consensus sequence (Tyr, Phe)-X-Cys-X.sub.(2-4)-Cys-X₃ -Phe-X₅ -Leu-X₂ -His-X.sub.(3-5) -His, wherein X represents amore variable amino acid. The cysteine and histidine residues coordinatea zinc ion, the three other conserved residues form a hydrophobic coreadjacent to the metal coordination unit, and the variable amino acidsmediate interactions with other molecules. The overall structureconsists of two antiparallel β-strands adjacent to an α-helix (Berg,supra). A protein may contain one or more zinc fingers which interactindependently of each other. In many instances, proteins which containzinc finger domains interact with specific double-stranded DNA (dsDNA)sequences, and carry out roles as transcription factors. Some zincfinger proteins, such as TFIIIA, bind to both dsDNA and tosingle-stranded RNA, while others, such as p43, appear to bind only tosingle-stranded 5S RNA (Berg, supra). Furthermore, certain zinc fingerproteins, including the human transcription factor SP1, bind DNA-RNAheteroduplexes with affinities comparable to or greater than those forDNA duplexes (Shi, Y. et al. (1995) Science 268:282-284).

A variant of the zinc finger described by a C₂ C₂ sequence motif isfound in the Xenopus G10 protein (McGrew, L. L. et al. (1989) Genes Dev.3: 803-815). G10 mRNA is a maternal transcript that is translationallyactivated during oocyte maturation. G10 protein consists of N-terminalcontaining a nuclear translocation signal (NTS) and alternating acidicand basic residues, and C-terminal sequence containing the C₂ C₂ -typezinc finger motif. G10 appears to function as a nuclear regulatoryprotein (McGrew et al., supra). Sequences highly homologous to G10 havebeen found in various organisms, including C. elegans, rice, and S.cerevisiae (Benit, P. et al. (1992) Yeast 8:147-153).

ZB domains which contain a C₃ HC₄ sequence motif are known as RINGdomains (Lovering, R. et al. (1993) Proc. Natl. Acad. Sci. USA90:2112-2116). The RING domain binds two zinc ions in an arrangementstructurally different from that of the zinc finger. The RING domainconsists of eight metal binding residues, and the sequences that bindthe two metal ions overlap (Barlow, P. N. et al. (1994) J. Mol. Biol.237:201-211). The consensus sequence C-X₂ -C-X.sub.(9-27) -C-X.sub.(1-3)-H-X.sub.(2-3) -C-X₂ -C-X.sub.(4-48) -C-X₂ -C provides for loops ofvarying length which form the overlapping Zn binding sites. The two Znbinding sites are formed by four pairs of metal-binding Cys and Hisresidues. The first and third pairs bind one metal ion, while the secondand fourth pairs bind the other (Barlow, et al., supra). Functions ofRING finger proteins are mediated through DNA binding and include theregulation of gene expression, DNA recombination, and DNA repair.

The murine BMI-1 gene encodes a protein of 324 amino acids. Thisprotein, which is found in the nuclei of a variety of normal cells,contains a RING domain near the aminoterminus (Haupt, Y. et al. (1991)Cell 65:753-763). Retroviral insertional mutagenesis of E-mu/myctransgenic mice by infection with Moloney murine leukemia virus (MuLV)accelerates development of B lymphoid tumors. In about half ofindependently induced pre-B-cell lymphomas, the provirus integrates inor near the BMI-1 gene, which results in enhanced transcription of thatgene. Haupt et al. (supra) concluded that myc-induced lymphomagenesismay entail the concerted action of several genes, including the putativenuclear regulator BMI-1. The human BMI-1 gene encodes a protein of 326amino acids which shares 98% identity to the amino acid sequence of themouse protein (Alkema, M. J. et al. (1993) Hum. Mol. Genet.2:1597-1603). Fluorescence in situ hybridization (FISH) on metaphasechromosome spreads localized the human BMI-1 proto-oncogene to the shortarm of chromosome 10 (10p13), a region known to be involved intranslocations in various leukemias (Alkema et al., supra).

The breast and ovarian cancer susceptibility-1 (BRCA1) gene encodes apredicted protein of 1,863 amino acids which contains a RING domain inthe amino-terminal region (Miki, Y. et al. (1994) Science 266:66-71).BRCA1 is expressed in numerous tissues, including breast and ovary. Insporadic breast cancer, BRCA1 mRNA levels are markedly decreased duringthe transition from carcinoma in situ to invasive cancer (Thompson M. E.et al. (1995) Nature Genet. 9:444-450). Furthermore, experimentalinhibition of BRCA1 expression with antisense oligonucleotides producedaccelerated growth of normal and malignant mammary cells, but had noeffect on nonmammary epithelial cells. Thompson et al. interpreted theseresults as an indication that BRCA1 may normally serve as a negativeregulator of mammary epithelial cell growth and that this function iscompromised in breast cancer either by direct mutation or by alterationsin gene expression.

A variation of the RING finger motif in which a His replaces the fourthCys of the consensus (C₃ HHC₃) is found in the protein product of theDrosophila developmental gene goliath (G1; Bouchard M. L. et al. (1993)Gene 125:205-209). The G1 gene is an abundant transcript of the visceralmesoderm of the Drosophila embryo. Mesoderm is one of the fundamentalembryonic germ layers which gives rise to internal structures such asthe body and gut musculature, fat body and heart. A high frequency ofhydrophobic and uncharged residues, primarily Ser, Gln and Pro (SQP-richregion), is found in the last one-third of the G1 protein. Based on theobservation that similar domains impart transcriptional activationability to eukaryotic DNA-binding proteins (Mitchell, P. J. et al.(1989) Science 245:371-378), Bouchard et al. suggest that the SQP-richregion of G1 is a potential transcriptional activation domain.

The discovery of polynucleotides encoding human zinc binding proteins,and the molecules themselves, provides a means to investigatephysiological processes relating to the control of cellulardifferentiation and proliferation under normal and disease conditions.Discovery of novel zinc binding proteins satisfies a need in the art byproviding new diagnostic or therapeutic compositions useful indiagnosing and treating diseases relating to disregulated cell growthand proliferation including cancer.

SUMMARY OF THE INVENTION

The present invention features three zinc binding proteins, designatedindividually as ZB-1, ZB-2 and ZB-3 and collectively as ZB, andcharacterized as having similarity to the zinc finger protein G10 andthe RING domain proteins BMI-1 and G1.

Accordingly, the invention features substantially purified ZB proteinsZB-1, ZB-2, and ZB-3 having the amino acid sequences shown in SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5, respectively.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode ZB proteins ZB-1, ZB-2, and ZB-3. In aparticular aspect, the polynucleotides are the nucleotide sequences ofSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, respectively.

The invention also features a polynucleotide sequence comprising thecomplement of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or variantsthereof. In addition, the invention features polynucleotide sequenceswhich hybridize under stringent conditions to SEQ ID NO:2, SEQ ID NO:4,or SEQ ID NO:6.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode ZB. The present inventionalso features antibodies which bind specifically to ZB, andpharmaceutical compositions comprising substantially purified ZB. Theinvention also features the use of agonists and antagonists of ZB.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) and nucleicacid sequence (SEQ ID NO:2) of ZB-1. The alignment was produced usingMACDNASIS PRO software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

FIG. 2A, 2B and 2C show the amino acid sequence (SEQ ID NO:3) andnucleic acid sequence (SEQ ID NO:4) of ZB-2.

FIGS. 3A, 3B and 3C show the amino acid sequence (SEQ ID NO:5) andnucleic acid sequence (SEQ ID NO:6) of ZB-3.

FIG. 4 shows the amino acid sequence alignment between ZB-1 (SEQ IDNO:1) and G10 protein from Xenopus laevis (GI 120625; SEQ ID NO:7). Thealignment was produced using the multisequence alignment program ofDNASTAR™ software (DNASTAR Inc, Madison Wis.).

FIG. 5 shows the amino acid sequence alignment between ZB-2 (SEQ IDNO:3) and human BMI-1 (GI 461632; SEQ ID NO:8).

FIG. 6 shows the amino acid sequence alignment between ZB-3 (SEQ IDNO:5) and Drosophila G1 protein (GI 157535; SEQ ID NO:9).

FIGS. 7A and 7B show the hydrophobicity plots (MACDNASIS PRO software)for ZB-1, SEQ ID NO:1 and G10 protein, SEQ ID NO:7; the positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity.

FIGS. 8A and 8B show the hydrophobicity plots for ZB-2, SEQ ID NO:3, andhuman BMI-1, SEQ ID NO:8.

FIGS. 9A and 9B show the hydrophobicity plot for ZB-3, SEQ ID NO:5, andG1 protein, SEQ ID NO:9.

FIGS. 10A, 10B and 10C show the northern analysis for SEQ ID NO:2. Thenorthern analysis was produced electronically using LIFESEQ™ database(Incyte Pharmaceuticals, Inc. Palo Alto, Calif.).

FIGS. 11A and 11B shows the northern analysis for SEQ ID NO:4.

FIGS. 12A, 12B and 12C show the northern analysis for SEQ ID NO:6.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

"Nucleic acid sequence", as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded and represent the sense or antisense strand. Similarly,"amino acid sequence", as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragments or portionsthereof, and to naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

ZB, as used herein, refers to the amino acid sequences of substantiallypurified ZB obtained from any species, particularly mammalian, includingbovine, ovine, porcine, murine, equine, and preferably human, from anysource whether natural, synthetic, semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEWfragment assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of ZB, as used herein, refers to an amino acid sequence thatis altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic ZB, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to ZB, causes a change in ZB which modulates the activity of ZB.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to ZB.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to ZB, blocks or modulates the biological orimmunological activity of ZB. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to ZB.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of ZB. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of ZB.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of ZB or portionsthereof and, as such, is able to effect some or all of the actions ofzinc binding protein-like molecules.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding ZB or the encoded ZB.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification", as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length human ZB-1 and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding ZB orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by northernanalysis is indicative of the presence of mRNA encoding ZB in a sampleand thereby correlates with expression of the transcript from thepolynucleotide encoding the protein.

"Alterations" in the polynucleotide of SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:6, as used herein, comprise any alteration in the sequence ofpolynucleotides encoding ZB including deletions, insertions, and pointmutations that may be detected using hybridization assays. Includedwithin this definition is the detection of alterations to the genomicDNA sequence which encodes ZB (e.g., by alterations in the pattern ofrestriction fragment length polymorphisms capable of hybridizing to SEQID NO:2, SEQ ID NO:4, or SEQ ID NO:6), the inability of a selectedfragment of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding ZB (e.g., using fluorescent in situhybridization (FISH) to metaphase chromosome spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind ZB polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide orpeptide used to immunize an animal can be derived from the translationof mRNA or synthesized chemically, and can be conjugated to a carrierprotein, if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin and thyroglobulin. The coupledpeptide is then used to immunize the animal (e.g., a mouse, a rat, or arabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

The invention is based on the discovery of novel human zinc bindingproteins (ZB-1, ZB-2, and ZB-3, collectively referred to as ZB), thepolynucleotides encoding ZB, and the use of these compositions for thediagnosis, prevention, or treatment of diseases related to disregulatedcell growth and proliferation including cancer.

Nucleic acids encoding the human ZB-1 of the present invention werefirst identified in Incyte Clone 3407, from a human leukemia-derivedmast cell line cDNA library (HMC1NOT01) through a computer-generatedsearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the following overlapping and/or extended nucleicacid sequences:Incyte Clones 3407 and 3664 (HMC1NOT01); 240102(HIPONOT01); 863306 (BRAITUT03); 913472 (STOMNOT02); and 1232134(LUNGFET03).

Nucleic acids encoding the human ZB-2 of the present invention werefirst identified in Incyte Clone 134194 from a bone marrow cDNA library(BMARNOT02) through a computer-generated search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:4, was derived from thefollowing overlapping and/or extended nucleic acid sequences:IncyteClones 1298467 (BRSTNOT07); 134194 (BMARNOT02); 280390 (LIVRNOT02); and879714 (THYRNOT02).

Nucleic acids encoding the human ZB-3 of the present invention werefirst identified in Incyte Clone 10773 from a human promonocyte THP-1cell line cDNA library (THP1PLB01) through a computer-generated searchfor amino acid sequence alignments. A consensus sequence, SEQ ID NO:6,was derived from the following overlapping and/or extended nucleic acidsequences:Incyte Clones 010773 (THP1PLB01); 159486 (ADENINB01); 477520and 520960 (MMLR2DT01); 562318 (NEUTLPT01); 568606 (MMLR3DT01); and741106 (PANCNOT04).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, and shown in FIGS. 1A and 1B.ZB-1 is 144 amino acids in length and contains a potential nucleartranslocation signal, consisting of predominantly basic residuesextending from position K₁ through K₉, followed by a region containing ahigh proportion of acidic residues from D₁₃ to E₃₈. As shown in FIG. 4,ZB-1 has chemical and structural homology with G10 protein from Xenopuslaevis (GI 120625; SEQ ID NO:7). In particular, ZB-1 shares 96% aminoacid sequence identity with Xenopus G10 protein, and the two proteinshave similar hydrophobicity profiles (FIGS. 7A and 7B). The C-terminusof ZB-1 contains a C₂ C₂ -type zinc finger domain spanning positionsC₁₀₁ to C₁₁₉. The presence in ZB-1 of a nuclear translation signal andthe zinc finger motif suggest a regulatory role in nuclear function.From the northern analysis (FIGS. 10A, 10B and 10C), ZB-1 is expressedin a variety of cell and tissue libraries. Of particular note is thehigh abundance of ZB-1 in hematopoietic tissues and cells involved inthe immune response and its presence in tumor-associated tissues andimmortalized cell lines. In addition, ZB-1 is found in several fetaltissue libraries and appears to have a role in fetal development.

In another embodiment, the invention encompasses the novel zinc bindingprotein ZB-2, a polypeptide comprising the amino acid sequence of SEQ IDNO:3, as shown in FIGS. 2A, 2B and 2C. ZB-2 is 180 amino acids inlength. As shown in FIG. 5, ZB-2 has chemical and structural homologywith human BMI-1 (GI 461632; SEQ ID NO:8). In particular, ZB-2 and BMI-1share 88% identity, and, as illustrated by FIGS. 8A and 8B, have rathersimilar hydrophobicity plots. ZB-2 contains a single RING domain,defined by amino acids C₂₇, C₃₀, C₄₂, H₄₄, C₄₇, C₅₀, C₆₄ and C₆₇, whichis precisely conserved among proteins involved in gene regulation andoncogenesis including BMI-1. Northern analysis (FIG. 11) reveals theexpression of ZB-2 sequence in approximately 50 cDNA libraries preparedfrom a wide variety of tissues, with highest abundance in adrenal gland,brain, thyroid, small intestine, lung, liver, prostate, colon, uterus,bladder, and bone marrow. Of particular note is the high abundance ofZB-2 in tissues relating to secretion or absorption, and its presence intumor-associated tissues and immortalized cell lines. In addition, ZB-2is found in a variety of fetal tissues and appears to have a role infetal development.

In an additional embodiment, the invention encompasses the novel zincbinding protein ZB-3, a polypeptide comprising the amino acid sequenceof SEQ ID NO:5, as shown in FIGS. 3A and 3B. ZB-3 is 276 amino acids inlength. As shown in FIG. 6, ZB-3 has chemical and structural homologywith Drosophila G1 protein (GI 157535; SEQ ID NO:9). In particular, ZB-3and G1 share 34% amino acid sequence identity, with maximal identity intheir N-terminal sequences. As illustrated by FIGS. 9A and 9B, ZB-3 andG1 have rather similar hydrophobicity plots. The single RING-like domainof ZB-3, defined by amino acids C₁₂₁, C₁₂₄, C₁₃₉, H₁₄₁, H₁₄₄, C₁₄₇,C₁₅₈, and C₁₆₁, is precisely conserved in G1. Northern analysis (FIGS.12A and 12B) shows the abundant expression of this sequence inhematopoietic cells involved in immune response, includingleukemia-derived promonocyte and mast cell lines, macrophages, andgranulocytes. ZB-3 encoding sequences are also expressed in glands andorgans involved in secretion and absorption, including breast, pinealgland, prostate, stomach, small intestine, bladder, liver, pancreas, andlung. Of particular note is the presence of ZB3 in tumor-associatedtissues and immortalized cell lines. In addition, ZB-3 is found in avariety of fetal tissues and appears to have a role in fetaldevelopment.

The invention also encompasses ZB variants. A preferred ZB variant isone having at least 80%, and more preferably 90%, amino acid sequencesimilarity to the ZB amino acid sequence (SEQ ID NO:1, SEQ ID NO:3, orSEQ ID NO:5). A most preferred ZB variant is one having at least 95%amino acid sequence similarity to SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:5.

The invention also encompasses polynucleotides which encode ZB.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of ZB can be used to generate recombinant molecules whichexpress ZB. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid of SEQ ID NO:2, SEQ ID NO:4,or SEQ ID NO:6 as shown in FIGS. 1A, 1B, 2A, 2B, 2C, 3A, 3B and 3C.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding ZB, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring ZB, and all such variations are to be considered asbeing specifically disclosed.

Although nucleotide sequences which encode ZB and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring ZB under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding ZB or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding ZB and its derivatives withoutaltering the encoded amino acid sequences include the production of RNAtranscripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode ZB and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding ZB or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,under various conditions of stringency. Hybridization conditions arebased on the melting temperature (Tm) of the nucleic acid bindingcomplex or probe, as taught in Wahl, G. M. and S. L. Berger (1987;Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-11), and may be used at a defined stringency.

Altered nucleic acid sequences encoding ZB which are encompassed by theinvention include deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent ZB. The encoded protein may also containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent ZB.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of ZB is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe gene encoding ZB. As used herein, an "allele" or "allelic sequence"is an alternative form of the gene which may result from at least onemutation in the nucleic acid sequence. Alleles may result in alteredmRNAs or polypeptides whose structure or function may or may not bealtered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, SEQUENASE Polymerase (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE amplification systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton MICRO LAB 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding ZB may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic.2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO®4.06 primer analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode ZB, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of ZB in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and express ZB.

As will be understood by those of skill in the art, it may beadvantageous to produce ZB-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter sequencesencoding ZB for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, or to introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding ZB may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of ZB activity, it may be useful to encode achimeric ZB protein that can be recognized by a commercially availableantibody. A fusion protein may also be engineered to contain a cleavagesite located between a sequence encoding ZB and the heterologous proteinsequence, so that ZB may be cleaved and purified away from theheterologous moiety.

In another embodiment, sequences encoding ZB may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of ZB, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of ZB, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active ZB, the nucleotide sequencesencoding ZB or functional equivalents, may be inserted into appropriateexpression vectors, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding ZB andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding ZB. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL), and the like, may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding ZB,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for ZB. For example, when largequantities of ZB are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding ZB may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding ZB may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S and 19S promoters of CaMV maybe used alone or in combination with the omega leader sequence from TMV(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoterssuch as the small subunit of RUBISCO or heat shock promoters may be used(Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105). These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.Such techniques are described in a number of generally available reviews(see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express ZB. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding ZB may be clonedinto a non-essential region of the virus, such as the polyhedrin gene,and placed under control of the polyhedrin promoter. Successfulinsertion of ZB will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. grugiperda cells or Trichoplusialarvae in which ZB may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding ZB may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing ZB in infected host cells (Logan, J. and Shenk, T.(1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding ZB. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding ZB, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express ZBmay be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding ZB isinserted within a marker gene sequence, recombinant cells containingsequences encoding ZB can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding ZB under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

Alternatively, host cells which contain sequences encoding andexpressing ZB may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA--DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

The presence of polynucleotide sequences encoding ZB can be detected byDNA--DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding ZB. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding ZB to detect transformantscontaining DNA or RNA encoding ZB. As used herein "oligonucleotides" or"oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression of ZB,using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson ZB is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding ZB includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, sequences encoding ZB, or anyportion thereof, may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits from Pharmacia & Upjohn (Kalamazoo, Mich.);Promega (Madison, Wis.); and U.S. Biochemical Corp. (Cleveland, Ohio).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding ZB may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeZB may be designed to contain signal sequences which direct secretion ofZB through a prokaryotic or eukaryotic cell membrane. Other recombinantconstructions may be used to join sequences encoding ZB to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and ZB may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingZB and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying ZB from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

In addition to recombinant production, fragments of ZB may be producedby direct peptide synthesis using solid-phase techniques (Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of ZB may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

THERAPEUTICS

Based on the chemical and structural homology between ZB-1 and XenopusG10, ZB-2 and human BMI-1, and ZB-3 and Drosophila G1, ZB appears toplay a role in cellular development and differentiation and may beinvolved in disorders relating to abnormal cell differentiation andproliferation including cancer. ZB is expressed in hematopoietic cells,brain, neuronal, and epithelial tissues, glands and tissues relating tosecretion or absorption, developing fetal tissues, and tissuesassociated with tumors and immortalized cell lines.

Therefore, in one embodiment, ZB or a fragment or derivative thereof maybe used to treat cells in vivo or ex vivo for the purposes of tissue ororgan regeneration. This embodiment would be of particular benefit inthe proliferation and differentiation of hematopoietic, nerve,epithelial or secretory cells.

In another embodiment, a vector capable of expressing ZB, or a fragmentor derivative thereof, may also be administered to a cell culture or asubject for ex vivo or in vivo therapy as described above.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding ZB may be administered to a subject to treat orprevent disorders which are associated with expression of ZB. Suchdisorders may include, but are not limited to, cancers of hematopoieticcells and tissues including leukemias, lymphomas, lymphosarcomas andmyelomas; cancers of brain and neuronal tissues including neuromas,neurogliomas, meningiomas, neuroblastomas and astrocytomas; cancers ofglands, tissues, and organs involved in secretion or absorption,including adrenal gland, thyroid, lung, pancreas, liver, prostate,uterus, bladder, kidney, testes, and the gastrointestinal tract (smallintestine, colon, rectum, and stomach); and other disorders relating toabnormal cellular differentiation, proliferation, or degeneration,including hyperaldosteronism, hypocortisolism (Addison's disease),hyperthyroidism (Grave's disease), hypothyroidism, colorectal polyps,gastritis, gastric and duodenal ulcers, ulcerative colitis, and Crohn'sdisease.

In another embodiment, antagonists or inhibitors of ZB may beadministered to a subject to treat or prevent any of the diseases ordisorders described above. In a particular aspect, antibodies which arespecific for ZB may be used directly as an antagonist, or indirectly asa targeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissues which express ZB.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, antisense sequences or vectors described above maybe administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

Antagonists or inhibitors of ZB may be produced using methods which aregenerally known in the art. In particular, purified ZB may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind ZB.

Antibodies which are specific for ZB may be generated using methods thatare well known in the art. Such antibodies may include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, Fabfragments, and fragments produced by a Fab expression library.Neutralizing antibodies, (i.e., those which inhibit dimer formation) areespecially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith ZB or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to ZB have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of ZB amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to ZB may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1985) Mol Cell Biol.62:109-120.

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-55; Neuberger, M. S. et al. (1984) Nature312:604-8; Takeda, S. et al. (1985) Nature 314:452-4). Alternatively,techniques described for the production of single chain antibodies maybe adapted, using methods known in the art, to produce ZB-specificsingle chain antibodies. Antibodies with related specificity, but ofdistinct idiotypic composition, may be generated by chain shuffling fromrandom combinatorial immunoglobulin libraries (Burton D. R. (1991) Proc.Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-37; Winter, G. et al. (1991) Nature 349:293-9).

Antibody fragments which contain specific binding sites for ZB may alsobe generated. For example, such fragments include, but are not limitedto, the F(ab')2 fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab')2 fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, W. D. et al. (1989) Science 254:1275-81).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between ZB and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering ZB epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encoding ZB,or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding ZB may be used in situations in which it would be desirable toblock the transcription of mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding ZB. Thus,antisense sequences may be used to modulate ZB activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligomers or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding ZB.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensepolynucleotides of the gene encoding ZB. These techniques are describedboth in Sambrook et al. (supra) and in Ausubel et al. (supra).

Genes encoding native ZB can be turned off by transforming a cell ortissue with expression vectors which express high levels of thepolynucleotide, or fragment thereof, which encodes ZB. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the genomic DNA,such vectors may continue to transcribe RNA molecules until they aredisabled by endogenous nucleases. Transient expression may last for amonth or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding ZB, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using "triple helix" base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding ZB.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding ZB. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of ZB, antibodies to ZB,mimetics, agonists, antagonists, or inhibitors of ZB. The compositionsmay be administered alone or in combination with at least one otheragent, such as stabilizing compound, which may be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffer saline. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of ZB, such labeling would include amount,frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example ZB or fragments thereof, antibodies of ZB,agonists, antagonists or inhibitors of ZB, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED50/LD50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind ZB may be usedfor the diagnosis of conditions or diseases characterized by expressionof ZB, or in assays to monitor patients being treated with ZB, agonists,antagonists or inhibitors. The antibodies useful for diagnostic purposesmay be prepared in the same manner as those described above fortherapeutics. Diagnostic assays for ZB include methods which utilize theantibody and a label to detect ZB in human body fluids or extracts ofcells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by joining them, either covalently ornon-covalently, with a reporter molecule. A wide variety of reportermolecules which are known in the art may be used, several of which aredescribed above.

A variety of protocols including ELISA, RIA, and FACS for measuring ZBare known in the art and provide a basis for diagnosing altered orabnormal levels of ZB expression. Normal or standard values for ZBexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toZB under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of ZB expressed in subjectsamples, control and disease from biopsied tissues are compared with thestandard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding ZBmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotide sequences, antisense RNA and DNA molecules,and PNAs. The polynucleotides may be used to detect and quantitate geneexpression in biopsied tissues in which expression of ZB may becorrelated with disease. The diagnostic assay may be used to distinguishbetween absence, presence, and excess expression of ZB, and to monitorregulation of ZB levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding ZB or closely related molecules, may be used to identifynucleic acid sequences which encode ZB. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding ZB, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe sequences encoding ZB. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequences ofSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 or from genomic sequenceincluding promoter, enhancer elements, and introns of the naturallyoccurring ZB.

Means for producing specific hybridization probes for DNAs encoding ZBinclude the cloning of nucleic acid sequences encoding ZB or ZBderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding ZB may be used for the diagnosis ofdisorders which are associated with expression of ZB. Examples of suchdisorders include cancers of hematopoietic cells and tissues includingleukemias, lymphomas, lymphosarcomas and myelomas; cancers of brain andneuronal tissues including neuromas, neurogliomas, meningiomas,neuroblastomas and astrocytomas; cancers of glands, tissues, and organsinvolved in secretion or absorption, including adrenal gland, thyroid,lung, pancreas, liver, prostate, uterus, bladder, kidney, and testes,and organs of the gastrointestinal tract including small intestine,colon, rectum, and stomach; other disorders relating to abnormalcellular differentiation, proliferation, or degeneration, includinghyperaldosteronism, hypocortisolism (Addison's disease), hyperthyroidism(Grave's disease), hypothyroidism, colorectal polyps, gastritis, gastricand duodenal ulcers, ulcerative colitis, and Crohn's disease. Thepolynucleotide sequences encoding ZB may be used in Southern or northernanalysis, dot blot, or other membrane-based technologies; in PCRtechnologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered ZB expression.Such qualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding ZB may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encoding ZBmay be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding ZB in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of ZB, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes ZB, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively low amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding ZB may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5'→3') and another with antisense(3'←5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of ZBinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequence whichencodes ZB may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequence may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma, R. S. et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding ZB on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, ZB, its catalytic or immunogenicfragments or oligopeptides thereof, can be used for screening librariesof compounds in any of a variety of drug screening techniques. Thefragment employed in such screening may be free in solution, affixed toa solid support, borne on a cell surface, or located intracellularly.The formation of binding complexes, between ZB and the agent beingtested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to ZB, large numbers of differentsmall test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The test compounds are reacted withZB, or fragments thereof, and washed. Bound ZB is then detected bymethods well known in the art. Purified ZB can also be coated directlyonto plates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding ZB specifically competewith a test compound for binding ZB. In this manner, the antibodies canbe used to detect the presence of any peptide which shares one or moreantigenic determinants with ZB.

In additional embodiments, the nucleotide sequences which encode ZB maybe used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I cDNA Library Construction

HMC1NOT01

The human mast cell HMCNOTO1 cDNA library was constructed by Stratageneusing mRNA purified from cultured HMC-1 cells. The cDNA library wasprepared by purifying mast cell poly(A+)RNA (mRNA) and thenenzymatically synthesizing double stranded complementary DNA (cDNA)copies of the mRNA. Synthetic adaptor oligonucleotides were ligated ontothe ends of the cDNA enabling its insertion into the lambda vector usingthe Uni-ZAP vector system (Stratagene).

BMARNOT02

Bone marrow poly (A+) RNA, derived from a pooled sample of bone marrowfrom the breast bones of 24 males and females whose ages ranged from 16to 70 years, was obtained from Clontech Laboratories Inc. (catalogue#6573-1 and #6573-2). The cDNA library was custom constructed byStratagene essentially as follows. cDNA synthesis was primed using botholigo d(T) and random hexamers, and the two cDNA libraries were treatedseparately. Synthetic adapter oligonucleotides were ligated onto cDNAends enabling its insertion into the Stratagene Uni-ZAP vector system.Finally, the two cDNA libraries were combined into a single library bymixing equal numbers of bacteriophage. The PBLUESCRIPT phagemid(Stratagene) was excised and transfected into E. coli host strainXL1-BLUE (Stratagene).

THP1PLB01

THP-1 is a human leukemic cell line derived from the blood of a1-year-old boy with acute monocytic leukemia. Cells used for the PMA+LPSlibrary (THP1PLB01) were cultured for 48 hr with 100 nm PMA in DMSO andfor 4 hr with 1 μg/ml LPS. The PMA+LPS-stimulated cells representactivated macrophages. The cDNA library was custom constructed byStratagene essentially as described below.

Stratagene prepared the cDNA library using oligo d(T) priming. Syntheticadapter oligonucleotides were ligated onto the cDNA molecules enablingthem to be inserted into the UNI-ZAP vector system (Stratagene). ThePBLUESCRIPT phagemid (Stratagene) was excised and transfected into E.coli host strain XL1-BLUE (Stratagene).

II Isolation and Sequencing of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which the host bacterial strain wasco-infected with both the library phage and an f1 helper phage. Enzymesderived from both the library-containing phage and the helper phagenicked the DNA, initiated new DNA synthesis from defined sequences onthe target DNA, and created a smaller, single stranded circular phagemidDNA molecule that included all DNA sequences of the PBLUESCRIPT phagemidand the cDNA insert. The phagemid DNA was released from the cells,purified, and used to reinfect fresh host cells (SOLR, Stratagene) wheredouble-stranded phagemid DNA was produced. Because the phagemid carriesthe gene for β-lactamase, the newly transformed bacteria were selectedon medium containing ampicillin.

Phagemid DNA was released from cells and purified using the Miniprep Kit(Cat. No. 77468; Advanced Genetic Technologies Corporation, GaithersburgMd.). This kit consists of a 96 well block with reagents for 960purifications. The recommended protocol was employed except for thefollowing changes: 1) the 96 wells were each filled with only 1 ml ofsterile Terrific Broth (Cat. No. 22711, LIFE TECHNOLOGIES™, GaithersburgMd.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteriawere cultured for 24 hours after the wells were inoculated and thenlysed with 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R at 2900 rpm for 5 min was performed before the contents ofthe block were added to the primary filter plate; and 4) the optionalstep of adding isopropanol to TRIS buffer was not routinely performed.After the last step in the protocol, samples were transferred to aBeckman 96-well block for storage.

Alternative methods of purifying phagemid DNA include the use of MAGICMINIPREPS™ DNA Purification System (Cat. No. A7100, Promega) orQIAWELL-8 Plasmid, QIAWELL PLUS DNA, and QIAWELL ULTRA DNA purificationsystems (Qiagen, Inc.).

The cDNAs were sequenced by the method of Sanger F. and A. R. Coulson(1975; J. Mol. Biol. 94:441f), using a Catalyst 800 (Perkin Elmer) orHamilton Micro Lab 2200 (Hamilton, Reno Nev.) in combination with fourPeltier Thermal Cyclers (PTC200 from MJ Research, Watertown Mass.) andApplied Biosystems 377 or 373 DNA Sequencing Systems (Perkin Elmer) andreading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in the GenBank and EMBL databasesusing two homology search algorithms. The first algorithm was originallydeveloped by Lipman D. J. and Pearson W. R. (1985; Science 227:1435). Inthis algorithm, the homologous regions are searched in a two-stepmanner. In the first step, highly homologous regions are determined bycalculating a matching score using a homology score table. In this step,the parameter "Ktup" is used to establish a shifting, minimum windowsize for comparing two sequences. Ktup also sets the number of basesthat must match to extract the highest homologous region among thesequences. In this step, no insertions or deletions are applied, and thehomology is displayed as an initial (INIT) value.

In the second step, the homologous regions are aligned to obtain thehighest matching score by inserting a gap when it is needed toaccommodate a probable deletion. The matching score obtained in thefirst step is recalculated using the homology score table and theinsertion score table to produce an optimized value.

DNA homologies between two sequences may also be examined graphicallyusing the Harr method of constructing dot matrix homology plots(Needleman, S. B. and Wunsch, C. O. (1970) J. Mol. Biol. 48:443). Thismethod produces a two-dimensional plot which can be useful indistinguishing between regions of homology and regions of repetition.

The second algorithm was developed by Applied Biosystems andincorporated into the INHERIT™ 670 sequence analysis system. In thisalgorithm, Pattern Specification Language (TRW Inc, Los Angeles, Calif.)was used to determine regions of homology. The three parameters thatdetermine how the sequence comparisons run were window size, windowoffset, and error tolerance. Using a combination of these threeparameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions of homology from chance matches. Smith-Waterman alignments wereused to display the results of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 sequence analysis system using the methods similar to thoseused in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were examined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding ZB occurs. Abundance and percent abundanceare also reported. Abundance directly reflects the number of times aparticular transcript is represented in a cDNA library, and percentabundance is abundance divided by the total number of sequences examinedin the cDNA library.

V Extension of Polynucleotides Encoding ZB to Full Length or to RecoverRegulatory Sequences

Polynucleotides encoding ZB (SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6)are used to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5' or 3', intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence outward, generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters:

    ______________________________________    Step 1       94° C. for 1 min (initial denaturation)    Step 2       65° C. for 1 min    Step 3       68° C. for 6 min    Step 4       94° C. for 15 sec    Step 5       65° C. for 1 min    Step 6       68° C. for 7 min    Step 7       Repeat step 4-6 for 15 additional cycles    Step 8       94° C. for 15 sec    Step 9       65° C. for 1 min    Step 10      68° C. for 7:15 min    Step 11      Repeat step 8-10 for 12 cycles    Step 12      72° C. for 8 min    Step 13      4° C. (and holding)    ______________________________________

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQUICK Kit (Qiagen Inc.). After recovery of the DNA, Klenowenzyme is used to trim single-stranded, nucleotide overhangs creatingblunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________    Step 1       940 C for 60 sec    Step 2       94° C. for 20 sec    Step 3       65° C. for 30 sec    Step 4       72° C. for 90 sec    Step 5       Repeat steps 2-4 for an additional 29 cycles    Step 6       72° C. for 180 sec    Step 7        4° C. (and holding)    ______________________________________

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although thelabeling of oligonucleotides, consisting of about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO 4.06 (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of -γ³² P!adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN®, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn).A portion containing 10⁷ counts per minute of each of the sense andantisense oligonucleotides is used in a typical membrane basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots, or after the blots areexposed to a Phosphoimager cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules to the sequence encoding ZB, or any part thereof, isused to inhibit in vivo or in vitro expression of naturally occurringZB. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on thesequences encoding ZB is used to inhibit expression of naturallyoccurring ZB. The complementary oligonucleotide is designed from themost unique 5' sequence as shown and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of a transcript encoding ZB bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5' sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,an effective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the signal or 5' codingsequence of the polypeptide as shown in FIGS. 1A, 1B, 2A, 2B, 2C, 3A, 3Band 3C.

VIII Expression of ZB

Expression of ZB is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, PSPORT, previously used for thegeneration of the cDNA library is used to express ZB in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion of ZBinto the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of ZB Activity

The binding of Zn²⁺ to ZB is assayed by monitoring the resulting changesin enthalpy (heat production or absorption) in an isothermal titrationmicrocalorimeter (Micro-Cal Inc., Northampton, Mass.). Titrationmicrocalorimetry measurements do not require labeling of the ligand orreceptor molecules; detection is based solely on the intrinsic change inthe heat of enthalpy upon binding. Multiple computer-controlledinjections of a known volume of ZnCl₂ solution are directed into athermally-controlled chamber containing ZB. The change in enthalpy aftereach injection is plotted against the number of injections to produce abinding isotherm. The volumes and concentrations of the injected ZnCl₂solution and of the ZB solution are used along with the binding isothermto calculate values for the number, affinity, and association constantof ZB with the Zn²⁺ ligand.

X Production of ZB Specific Antibodies

ZB that is substantially purified using PAGE electrophoresis (Sambrook,supra), or other purification techniques, is used to immunize rabbitsand to produce antibodies using standard protocols. The amino acidsequence deduced from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 isanalyzed using DNASTAR software (DNASTAR Inc.) to determine regions ofhigh immunogenicity and a corresponding oligopolypeptide is synthesizedand used to raise antibodies by means known to those of skill in theart. Selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions, is described by Ausubel et al.(supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring ZB Using Specific Antibodies

Naturally occurring or recombinant ZB is substantially purified byimmunoaffinity chromatography using antibodies specific for ZB. Animmunoaffinity column is constructed by covalently coupling ZB antibodyto an activated chromatographic resin, such as CnBr-activated Sepharose(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

Media containing ZB is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof ZB (e.g., high ionic strength buffers in the presence of detergent).The column is eluted under conditions that disrupt antibody/ZB binding(eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such asurea or thiocyanate ion), and ZB is collected.

XII Identification of Molecules Which Interact with ZB

ZB or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J.133:529-39). Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled ZB, washed and any wellswith labeled ZB complex are assayed. Data obtained using differentconcentrations of ZB are used to calculate values for the number,affinity, and association of ZB with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 9    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 144 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetProLysValLysArgSerArgLysAlaProProAspGlyTrpGlu    151015    LeuIleGluProThrLeuAspGluLeuAspGlnLysMetArgGluAla    202530    GluThrGluProHisGluGlyLysArgLysValGluSerLeuTrpPro    354045    IlePheArgIleHisHisGlnLysThrArgTyrIlePheAspLeuPhe    505560    TyrLysArgLysAlaIleSerArgGluLeuTyrGluTyrCysIleLys    65707580    GluGlyTyrAlaAspLysAsnLeuIleAlaLysTrpLysLysGlnGly    859095    TyrGluAsnLeuCysCysLeuArgCysIleGlnThrArgAspThrAsn    100105110    PheGlyThrAsnCysIleCysArgValProLysSerLysLeuGluVal    115120125    GlyArgIleIleGluCysThrHisCysGlyCysArgGlyCysSerGly    130135140    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 828 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GCCTGAAGAGCGGAAGCCTTCTGTCGAGAAGCAGCTACCCAAGCTCCAGGAGCTTCCGAA60    GAAACAGGACCAGAGAGGGAAGGTGACCTGAAAGTCACAGAATAATTTTTTAGAGCTGAA120    CAAGAATCCAAGCCTGCAACTGCAGAGACGAGAGATCTTTCTGCTGTCTATACTCTTGGA180    AAGCACATCCTAAGATCTTTGCAGATTATCCTGTGGAAGGAAAATGCCTAAAGTCAAAAG240    AAGCCGGAAAGCACCCCCAGATGGCTGGGAGTTGATTGAGCCAACACTGGATGAATTAGA300    TCAAAAGATGAGAGAAGCTGAAACAGAACCGCATGAGGGAAAGAGGAAAGTGGAATCTCT360    GTGGCCCATCTTCAGGATCCACCACCAGAAAACCCGCTACATCTTCGACCTCTTTTACAA420    GCGGAAAGCCATCAGCAGAGAACTCTATGAATATTGTATTAAAGAAGGCTATGCAGACAA480    AAACCTGATTGCAAAATGGAAAAAGCAAGGATATGAGAACTTGTGCTGCCTGCGGTGCAT540    TCAGACACGGGACACCAACTTCGGGACGAACTGCATCTGCCGCGTGCCCAAAAGCAAGCT600    GGAAGTGGGCCGCATCATCGAGTGCACACACTGTGGCTGTCGTGGCTGCTCTGGCTGAGG660    CTGGCGCGCTCCACCCTGGACTCTGGACTTCGCAGGTTCCTGCCTGTCACGCCACCCCCT720    TCCTGGGAGCAGCGAGCAGTGCCCCAGGCCCGAGTTGGAGCACGGTCTCTATGGGGAAGG780    CTTCGCTGTCTATCAGCTGTGATTTGTAAAAATAAAATCTTTAAATCT828    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 180 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    MetAlaAlaAlaGluGluGluAspGlyGlyProGluGlyProAsnArg    151015    GluArgGlyGlyAlaGlyAlaThrPheGluCysAsnIleCysLeuGlu    202530    ThrAlaArgGluAlaValValSerValCysGlyHisLeuTyrCysTrp    354045    ProCysLeuHisGlnTrpLeuGluThrArgProGluArgGlnGluCys    505560    ProValCysLysAlaGlyIleSerArgGluLysValValProLeuTyr    65707580    GlyArgGlySerGlnLysProGlnAspProArgLeuLysThrProPro    859095    ArgProGlnGlyGlnArgProAlaProGluSerArgGlyGlyPheGln    100105110    ProPheGlyAspThrGlyGlyPheHisPheSerPheGlyValGlyAla    115120125    PheProPheGlyPhePheThrThrValPheAsnAlaHisGluProPhe    130135140    ArgArgGlyThrGlyValAspLeuGlyGlnGlyHisProAlaSerSer    145150155160    TrpGlnAspSerLeuPheLeuPheLeuAlaIlePhePhePhePheTrp    165170175    LeuLeuSerIle    180    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 944 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CAACGATCGTGGGCAGGAGGTGGTTTCTGGTTTGTTGGGGCGTGTGTATGTGTATTTGGG60    GGGACTGAAGGGTACGTGGGGCGAAACAAAACCGGCCATGGCAGCAGCGGAGGAGGAGGA120    CGGGGGCCCCGAAGGGCCAAATCGCGAGCGGGGCGGGGCGGGCGCGACCTTCGAATGTAA180    TATATGTTTGGAGACTGCTCGGGAAGCTGTGGTCAGTGTGTGTGGCCACCTGTACTGTTG240    GCCATGTCTTCATCAGTGGCTGGAGACACGGCCAGAACGGCAAGAGTGTCCAGTATGTAA300    AGCTGGGATCAGCAGAGAGAAGGTTGTCCCGCTTTATGGGCGAGGGAGCCAGAAGCCCCA360    GGATCCCAGATTAAAAACTCCACCCCGCCCCCAGGGCCAGAGACCAGCTCCGGAGAGCAG420    AGGGGGATTCCAGCCATTTGGTGATACCGGGGGCTTCCACTTCTCATTTGGTGTTGGTGC480    TTTTCCCTTTGGCTTTTTCACCACCGTCTTCAATGCCCATGAGCCTTTCCGCCGGGGTAC540    AGGTGTGGATCTGGGACAGGGTCACCCAGCCTCCAGCTGGCAGGATTCCCTCTTCCTGTT600    TCTCGCCATCTTCTTCTTTTTTTGGCTGCTCAGTATTTGAGCTATGTCTGCTTCCTGCCC660    ACCTCCAGCCAGAGAAGAATCAGTATATTGAAGGTCCCTGCTGAMCCTTCCGTATCCTGG720    AACCCCTGACCCTCTTTTTTTTTTGCTAANGGCACCCTGAACTTTTCCNGAAGGCTGGGA780    AAAAATTAATCTTTCTTAATGGAAANCTCTCCCCAAGNCCTCATAACTTTTTAATCCCCC840    CNGGGAAGAGATGAATAAAAAATTNTTCNCCCCCAATTTTGCTTCCCGATTCTNATTNAC900    TCAAGTGGCAATTCCCTNATCTCCCCTCCACTTTGATAATTATT944    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 276 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    MetThrHisProGlyThrGlyAspIleIleAlaValMetIleThrGlu    151015    LeuArgGlyLysAspIleLeuSerTyrLeuGluLysAsnIleSerVal    202530    GlnMetThrIleAlaValGlyThrArgMetProProLysAsnPheSer    354045    ArgGlySerLeuValPheValSerIleSerPheIleValLeuMetIle    505560    IleSerSerAlaTrpLeuIlePheTyrPheIleGlnLysIleArgTyr    65707580    ThrAsnAlaArgAspArgAsnGlnArgArgLeuGlyAspAlaAlaLys    859095    LysAlaIleSerLysLeuThrThrArgThrValLysLysGlyAspLys    100105110    GluThrAspProAspPheAspHisCysAlaValCysIleGluSerTyr    115120125    LysGlnAsnAspValValArgIleLeuProCysLysHisValPheHis    130135140    LysSerCysValAspProTrpLeuSerGluHisCysThrCysProMet    145150155160    CysLysLeuAsnIleLeuLysAlaLeuGlyIleValProAsnLeuPro    165170175    CysThrAspAsnValAlaPheAspMetGluArgLeuThrArgThrGln    180185190    AlaValAsnArgArgSerAlaLeuGlyAspLeuAlaGlyAspAsnSer    195200205    LeuGlyLeuGluProLeuArgThrSerGlyIleSerProLeuProGln    210215220    AspGlyGluLeuThrProArgThrGlyGluIleAsnIleAlaValThr    225230235240    LysGluTrpPheIleIleAlaSerPheGlyLeuLeuSerAlaLeuThr    245250255    LeuCysTyrMetIleIleArgAlaThrAlaSerLeuAsnAlaAsnGlu    260265270    ValGluTrpPhe    275    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1253 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GNCGCTAACGGGCTTGANTCCCCCAAGGCCGAGGTCCGCGGCCAGGTGCTGGCGCCGCTG60    CCCCTCCACGGAGTTGCTGATCATCTGGGCTGTGATCCACAAACCCGGTTCTTTGTCCCT120    CCTAATATCAAACAGTGGATTGCCTTGCTGCAGAGGGGAAACTGCACGTTTAAAGAGAAA180    ATATCACGGGCCGCTTTCCACAATGCAGTTGCTGTAGTCATCTACAATAATAAATCCAAA240    GAGGAGCCAGTTACCATGACTCATCCAGGCACTGGAGATATTATTGCTGTCATGATAACA300    GAATTGAGGGGTAAGGATATTTTGAGTTATCTGGAGAAAAACATCTCTGTACAAATGACA360    ATAGCTGTTGGAACTCGAATGCCACCGAAGAACTTCAGCCGTGGCTCTCTAGTCTTCGTG420    TCAATATCCTTTATTGTTTTGATGATTATTTCTTCAGCATGGCTCATATTCTACTTCATT480    CAGAAGATCAGGTACACAAATGCACGCGACAGGAACCAGCGTCGTCTCGGAGATGCAGCC540    AAGAAAGCCATCAGTAAATTGACAACCAGGACAGTAAAGAAGGGTGACAAGGAAACTGAC600    CCAGACTTTGATCATTGTGCAGTCTGCATAGAGAGCTATAAGCAGAATGATGTCGTCCGA660    ATTCTCCCCTGCAAGCATGTTTTCCACAAATCCTGCGTGGATCCCTGGCTTAGTGAACAT720    TGTACCTGTCCTATGTGCAAACTTAATATATTGAAGGCCCTGGGAATTGTGCCGAATTTG780    CCATGTACTGATAACGTAGCATTCGATATGGAAAGGCTCACCAGAACCCAAGCTGTTAAC840    CGAAGATCAGCCCTCGGCGACCTCGCCGGCGACAACTCCCTTGGCCTTGAGCCACTTCGA900    ACTTCGGGGATCTCACCTCTTCCTCAGGATGGGGAGCTCACTCCGAGAACAGGAGAAATC960    AACATTGCAGTAACAAAAGAATGGTTTATTATTGCCAGTTTTGGCCTCCTCAGTGCCCTC1020    ACACTCTGCTACATGATCATCAGAGCCACAGCTAGCTTGAATGCTAATGAGGTAGAATGG1080    TTTTGAAGAAGAAAAAACCTGCTTTCTGACTGATTTTGCCTTGAAGGAAAAAAGAACCTA1140    TTTTTGTGCATCATTTACCAATCATGCCACACAAGCATTTATTTTTAGTACATTTTATTT1200    TTTCATAAAATTGCTAATGCCAAAGGTTTGTATTAAAAGGGATAAATAGTAAA1253    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 144 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: GenBank    (B) CLONE: 120625    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    MetProLysValLysArgSerArgLysProProProAspGlyTrpGlu    151015    LeuIleGluProThrLeuAspGluLeuAspGlnLysMetArgGluAla    202530    GluThrAspProHisGluGlyLysArgLysValGluSerLeuTrpPro    354045    IlePheArgIleHisHisGlnLysThrArgTyrIlePheAspLeuPhe    505560    TyrLysArgLysAlaIleSerArgGluLeuTyrAspTyrCysIleArg    65707580    GluGlyTyrAlaAspLysAsnLeuIleAlaLysTrpLysLysGlnGly    859095    TyrGluAsnLeuCysCysLeuArgCysIleGlnThrArgAspThrAsn    100105110    PheGlyThrAsnCysIleCysArgValProLysThrLysLeuGluVal    115120125    GlyArgIleIleGluCysThrHisCysGlyCysArgGlyCysSerGly    130135140    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 326 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: GenBank    (B) CLONE: 461632    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetHisArgThrThrArgIleLysIleThrGluLeuAsnProHisLeu    151015    MetCysValLeuCysGlyGlyTyrPheIleAspAlaThrThrIleIle    202530    GluCysLeuHisSerPheCysLysThrCysIleValArgTyrLeuGlu    354045    ThrSerLysTyrCysProIleCysAspValGlnValHisLysThrArg    505560    ProLeuLeuAsnIleArgSerAspLysThrLeuGlnAspIleValTyr    65707580    LysLeuValProGlyLeuPheLysAsnGluMetLysArgArgArgAsp    859095    PheTyrAlaAlaHisProSerAlaAspAlaAlaAsnGlySerAsnGlu    100105110    AspArgGlyGluValAlaAspGluAspLysArgIleIleThrAspAsp    115120125    GluIleIleSerLeuSerIleGluPhePheAspGlnAsnArgLeuAsp    130135140    ArgLysValAsnLysAspLysGluLysSerLysGluGluValAsnAsp    145150155160    LysArgTyrLeuArgCysProAlaAlaMetThrValMetHisLeuArg    165170175    LysPheLeuArgSerLysMetAspIleProAsnThrPheGlnIleAsp    180185190    ValMetTyrGluGluGluProLeuLysAspTyrTyrThrLeuMetAsp    195200205    IleAlaTyrIleTyrThrTrpArgArgAsnGlyProLeuProLeuLys    210215220    TyrArgValArgProThrCysLysArgMetLysIleSerHisGlnArg    225230235240    AspGlyLeuThrAsnAlaGlyGluLeuGluSerAspSerGlySerAsp    245250255    LysAlaAsnSerProAlaGlyGlyValProSerThrSerSerCysLeu    260265270    ProSerProSerThrProValGlnSerProHisProGlnPheProHis    275280285    IleSerSerThrMetAsnGlyThrSerAsnSerProSerGlyAsnHis    290295300    GlnSerSerPheAlaAsnArgProArgLysSerSerValAsnGlySer    305310315320    SerAlaThrSerSerGly    325    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 284 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (vii) IMMEDIATE SOURCE:    (A) LIBRARY: GenBank    (B) CLONE: 157535    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    MetGlnLeuGluLysMetGlnIleLysGlyLysThrArgAsnIleAla    151015    AlaValIleThrTyrGlnAsnIleGlyGlnAspLeuSerLeuThrLeu    202530    AspLysGlyTyrAsnValThrIleSerIleIleGluGlyArgArgGly    354045    ValArgThrIleSerSerLeuAsnArgThrSerValLeuPheValSer    505560    IleSerPheIleValAspAspIleLeuCysTrpLeuIlePheTyrTyr    65707580    IleGlnArgPheArgTyrMetGlnAlaLysAspGlnGlnSerArgAsn    859095    LeuCysSerValThrLysLysAlaIleMetLysIleProThrLysThr    100105110    GlyLysPheSerAspGluLysAspLeuAspSerAspCysCysAlaIle    115120125    CysIleGluAlaTyrLysProThrAspThrIleArgIleLeuProCys    130135140    LysHisGluPheHisLysAsnCysIleAspProTrpLeuIleGluHis    145150155160    ArgThrCysProMetCysLysLeuAspValLeuLysPheTyrGlyTyr    165170175    ValValGlyAspGlnIleTyrGlnThrProSerProGlnHisThrAla    180185190    ProIleAlaSerIleGluGluValProValIleValValAlaValPro    195200205    HisGlyProGlnProLeuGlnProLeuGlnAlaSerAsnMetSerSer    210215220    PheAlaProSerHisTyrPheGlnSerSerArgSerProSerSerSer    225230235240    ValGlnGlnGlnLeuAlaProLeuThrTyrGlnProHisProGlnGln    245250255    AlaAlaSerGluArgGlyArgArgAsnSerAlaProAlaThrMetPro    260265270    HisAlaIleThrAlaSerHisGlnValThrAspVal    275280    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ IDNO:1.
 2. A hybridization probe consisting of the polynucleotide sequenceof claim
 1. 3. An isolated and purified polynucleotide sequenceconsisting of SEQ ID NO:2.
 4. An isolated and purified polynucleotidesequence which is complementary to the polynucleotide sequence ofclaim
 1. 5. A hybridization probe consisting of the polynucleotidesequence of claim
 4. 6. An expression vector containing thepolynucleotide sequence of claim
 1. 7. A host cell containing theexpression vector of claim
 6. 8. A method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, the method comprisingthe steps of:a) culturing the host cell of claim 7 under conditionssuitable for the expression of the polypeptide; and b) recovering thepolypeptide from the host cell culture.